The A→G mutation at the 3243rd position of mitochondrial DNA (mt3243) is a mutation existing in 1% of Japanese diabetes mellitus patients, and occurs at the highest frequency among diabetic conditions caused by abnormality of a single gene. One of the characteristics of abnormality of mitochondrial genes is coexistence of normal and abnormal mitochondrial DNAs in various proportions, and this state is referred to as heteroplasmy. The ratio of mt3243 mutation heteroplasmy is said to relate to progression degree of the pathological condition, and it is considered that it can be used for the determination of progression degree of the pathological condition or therapeutic effect.
If the mt3243 mutation exists, a recognition site of a restriction enzyme emerges at the position of the mutation. Therefore, the mutation can be detected by a method of amplifying DNA by PCR so that a portion including the mutation position should be amplified, digesting the amplification product with a restriction enzyme and determining whether the DNA has been digested or not by electrophoresis (PCR-RFLP) (for example, refer to The Japanese Journal of Clinical Pathology, vol. 44, 8, pp. 778-782, 1996).
Because PCR amplifies templates of several molecules several billion times, even a trace amount of contaminant may cause a false positive or false negative result. In PCR-RFLP, the amplification product needs to be collected and subjected to a treatment with a restriction enzyme after PCR, and therefore the amplification product may contaminate the subsequent reaction system. Accordingly, a false positive or false negative result may be obtained.
Further, DNA is treated with a restriction enzyme and then subjected to electrophoresis after completion of PCR. Therefore, time required for the detection becomes extremely long. In addition, because the procedure is complicated, automatization is difficult. Furthermore, because denaturation and annealing are repeated during PCR, a normal type sequence and a mutant type sequence may erroneously bind to each other. Such a product is not recognized by a restriction enzyme, and therefore it is not digested (DNA is digested by a restriction enzyme only when both of the double strands are of mutant type). Accordingly, in the quantification of the ratio of heteroplasmy, the proportion of the mutant type becomes lower than the actual value.
Meanwhile, as an allele specific amplification method, a method called MASA (mutant allele specific amplification) method is known (for example, refer to Sekiya T. et al. ed., “PCR Front Line—From Basic Techniques To Applications”, Kyoritsu Shuppan, pp. 140-142, 1997). In this method, the mutation allele is specifically amplified by performing PCR using a primer pair designed so that the 3′ end of one primer should be a mutated nucleotide.
Further, a method is also known in which by using a system in which fluorescence changes depending on amount of an amplification product; PCR amplification product is quantified on a real time basis by measuring fluorescence; and on the basis of the results, nucleic acids in a sample are quantified (real time quantitative PCR). By performing the MASA method in accordance with this method, an amplification product obtained by the MASA method can be quantified.
Furthermore, a method is generally known in which a region containing a mutation is amplified by PCR, then a melting curve analysis is performed by using a nucleic acid probe labeled with a fluorescent dye, and the mutation is analyzed on the basis of the result of the melting curve analysis (Clinical Chemistry, vol. 46, 5, pp. 631-635, 2000; Japanese Patent Application Laid-open (Kokai) No. 2002-119291).